Hemin upregulates Egr-1 expression in vascular smooth muscle cells via reactive oxygen species ERK-1/2-Elk-1 and NF-kappaB.

Reactive oxygen species (ROS) and oxidant stress are important mediators of cardiovascular pathologies including atherosclerosis. One source of ROS in the vasculature is free heme released from hemoglobin. Because Egr-1, the regulator of cell proliferation and apoptosis, is also induced by oxidant stress and is likewise implicated in atherosclerosis, we examined the regulation of Egr-1 by heme in vascular smooth muscle cells (SMCs). Hemin increased Egr-1 expression (mRNA, protein) within 30 minutes and ERK-1/2 phosphorylation and nuclear translocation within 5 minutes. Inhibiting hemin-induced ERK-1/2 activation by U0126 (MAPK-inhibitor), the antioxidant N-acetyl cysteine, the NADPH oxidase inhibitors apocynin and diphenyleneiodonium chloride, the superoxide scavenger tiron, or tricarbonyldichlororuthenium(II)-dimer (carbon-monoxide donor; CORM-2) blocked hemin-induced Egr-1 expression. Hemin activated Elk-1, SRF, and NF-kappaB and promoted their interaction with the Egr-1 promoter. Downregulating Elk-1 (via siRNA) or blocking NF-kappaB activation (via BAY-11-7082) abolished hemin induction of Egr-1. Finally, hemin-induced Egr-1 bound the promoters of tissue factor (TF), Plasminogen Activator Inhibitor (PAI)-1, and NGF-1A Binding (NAB)-2, upregulating their expression, and increased the biochemical activity of TF and PAI-1. Upregulation of Egr-1 and its target genes by heme-induced oxidant stress may be an important event in the initiation and progression of inflammatory vascular diseases such as atherosclerosis.

Differential regulation of early growth response gene-1 expression by insulin and glucose in vascular endothelial cells.

OBJECTIVE: Early growth response gene (Egr)-1 is a key transcription factor involved in vascular pathophysiology. Its role in diabetic vascular complications, however, remains unclear. Because hyperinsulinemia and hyperglycemia are major risk factors leading to diabetic vascular complications, we examined the effect of insulin and glucose on Egr-1 expression in murine glomerular vascular endothelial cells. METHODS AND RESULTS: Insulin or glucose, when added separately, increased egr-1 mRNA levels and promoter activity, as well as Egr-1 protein levels in nuclear extracts. When insulin was added to cells preincubated with glucose, the two had an additive effect on Egr-1 expression. Furthermore, vascular endothelial growth factor receptor-1 (flt-1) and plasminogen activator inhibitor-1, two known Egr-1-responsive genes, were also upregulated in the presence of insulin or glucose. An investigation into the underlying molecular mechanisms demonstrated that insulin, but not glucose, increased Egr-1 expression through extracellular signal-regulated kinase 1/2 activation, which is consistent with our previous reports. In contrast, inhibition of protein kinase C by phorbol ester or by the specific protein kinase C inhibitor chelerythrine chloride downregulated glucose-induced, but not insulin-induced, Egr-1 expression. CONCLUSIONS: Differential regulation of Egr-1 expression by insulin and glucose in vascular cells may be one of the initial key events that plays a crucial role in the development of diabetic vascular complications.